Liposomes are described quite widely in the literature and their structure is well known. Liposomes are unilamellar or multilamellar lipid vesicles which enclose a fluid space or spaces. The walls of the vesicles are formed by a bimolecular layer of one or more lipid components having polar heads and non-polar tails. In an aqueous (or polar) solution, the polar heads of one layer orient outwardly to extend into the surrounding medium, and the non-polar tail portions of the lipid associate with each other, thus providing a polar surface and non-polar core in the wall of the vesicle. Unilamellar liposomes have one such bimolecular layer, whereas multilamellar liposomes generally have a plurality of substantially concentric bimolecular layers.
A variety of methods for preparing liposomes are known, many of which have been described by Szoka and Papahadjopoulos, Ann. Rev. Biophysics Bioeng. 9: 467-508 (1980) and in Liposome Technology, Preparation of Liposomes, Vol I, Gregoriadis (Ed.), CRC Press, Inc. (1984). Also, several liposome encapsulation methods are disclosed in the patent literature, notably in U.S. Pat. No. 4,235,871, issued to Papahadjopoulos et al. on Nov. 25, 1980, and in U.S. Pat. No. 4,016,100, issued to Suzuki et al. on Apr. 5, 1977.
In order for liposomes to be useful in commercial settings, it is desirable to extend the shelf-life of liposomal preparations. Such preparations must have long enough shelf-lives to allow them to be easily manufactured, shipped and stored by intermediate and ultimate users under a variety of temperature conditions. With particular regard to the pharmaceutical industry, it is important to be able to store liposomal preparations for long periods of time without incurring substantial leakage of the incorporated drug.
Liposomal stability on storage is defined generally as the extent to which a given preparation retains both its original structure and size distribution and if applicable, its load of incorporated agent, whether therapeutic or diagnostic in nature. Instability can occur, for example, when vesicle size increases spontaneously upon standing as a result of fusion of colliding vesicles. The larger vesicles will exhibit drastically different pharmacokinetics in vivo because their size determines their clearance rates and tissue distribution; for instance, large liposomes are removed from the circulation more rapidly than smaller ones. In addition, liposomes in an aqueous liposome dispersion can aggregate and precipitate as a sediment. Although such sediments can usually be re-dispersed, the structure and size distribution of the original dispersion may be changed. Finally, another important factor with regard to instability is that incorporated substances of low molecular weight are likely to leak from stored liposomes. See generally G. Gregoriadis, Liposomes for Drugs and Vaccines in 3 Trends in Biotechnology, 235-241 (1985). If the content of the incorporated agent is small and/or the volume of the external aqueous medium is large, such leakage can represent a significant proportion of the total content of the agent in the liposomes.
Research directed to prolonging liposomal stability on storage has focused on liposome preservation in the form of lyophilization. Lyophilization refers to the process whereby a substance is prepared in dry form by rapid freezing and dehydration under high vacuum. Traditional wisdom dictates that phospholipid vesicles cannot be lyophilized successfully. Recent studies done by Drs. John and Lois Crowe at the University of California at Davis indicate that the disaccharide, trehalose, functions as a cryoprotectant during lyophilization and the studies conclude that optimal results are achieved when the cryoprotectant is located inside as well as outside the liposome. L. M. Crowe, et al., 1 Archives of Biochemistry and Physics 242 (1985). See also J. H. Crowe, L. M. Crowe, Cryobiology, 19, 317 (1982) In Biological Membranes, D. Chapman, Ed. (Academic Press, N.Y. 5, 57) in which it was reported that certain organisms such as nematodes, were able to survive dehydration in the presence of trehalose. Battelle Memorial Institute, Basel, has also disclosed the use of proteins and polysaccharides as liposome preservation agents during lyophilization, resulting in reported undamaged liposome levels of only approximately 70%. Schneider, et al., Process for the Dehydration of a Colloidal Dispersion of Liposomes, U.S. Pat. No. 4,229,360 (Oct. 21, 1980). Several other patents have been issued which disclose various methods to preserve liposomes utilizing lyophilization techniques. Evans, et al., Process for Preparing Freeze-Dried Liposome Compositions, U.S. Pat. No. 4,370,349 (Jan. 25, 1983); Weiner, et al., Storage-Stable Lipid Vesicles and Method of Preparation, U.S. Pat. No. 4,397,846 (Aug. 9, 1983); Vanlerberghe, et al., Storage Stability of Aqueous Dispersions of Spherules (Jan. 27, 1981). The stabilizing effect of sugars on sarcoplasmic reticulum subjected to freeze-drying and rehydration, and on microsomes and egg phosphatidylcholine SUV subjected to freeze-thawing have also previously been noted.
Lyophilization is an expensive procedure and would require considerable plant investment in order to produce dehydrated liposomal preparations on a commercial scale. Spray-drying and scrape surface drying (drum-drying) techniques, described generally in Hansen, U.S. Pat. No. 3,549,382 (Dec. 22, 1970), are less expensive to utilize commercially. Moreover, such techniques require the use of less energy than does the lyophilizing technique. Thin film evaporation constitutes an equivalent technology to spray-drying and scrape surface drying. Each of these three techniques is capable of causing flash evaporation, or rapid vaporization of a dispersion medium without damaging the integrity of the materials, in the present case liposomes, suspended in that medium. Such vaporization occurs in a temperature range of about 60.degree. C. to about 150.degree. C.
Recently unilamellar lipid vesicles have become important in several research areas dealing with membrane mediated processes such as membrane fusion, interfacial catalysis, energy conduction and conversion, drug delivery and targeting. There is hope that this kind of research will eventually lead to industrial applications of unilamellar lipid vesicles. In any practical application the questions of long-term storage and related to it vesicle and bilayer stability are important. It is well-known that aqueous dispersions of small unilamellar lipid vesicles (SUV) are thermodynamically unstable. For instance, SUV made of zwitterionic phosphatidylcholines tend to aggregate and/or fuse to large multilamellar lipid particles at room temperature. Furthermore, they undergo chemical degradation with time. The process of fusion of SUV is greatly accelerated when SUV are subjected to freeze-thawing or dehydration. It has been shown that SUV of egg phosphatidylcholine revert to large multilamellar structures upon freezing and thawing. G. Strauss and H. Hauser, Proc. Natl. Acad. Sci. USA 83, 2422 (1986), the disclosure of which is incorporated herein by reference. SUV are therefore an ideal system to test the stabilizing effect of various additives and to test dehydration by spray-drying or an equivalent technology.